Multiplexed imaging using strand displacement

ABSTRACT

The present disclosure describes various improved methods for imaging at least one target in a sample, including methods employing an adapter strand oligonucleotide and a bridge strand oligonucleotide. Some methods also employ bouncer oligonucleotides and/or blocker oligonucleotides. Some methods also use two partial docking strands to detect proximity of the partial docking strands to each other.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/117,194, filed Aug. 30, 2018, which is a continuation ofInternational Application No. PCT/US2017/032398 filed May 12, 2017, andclaims the benefit of priority of US Provisional Application Nos.62/336,676, filed May 15, 2016, and 62/343,791, filed May 31, 2016, thecontents of all of which are incorporated by reference herein in theirentirety for any purpose.

DETAILED DESCRIPTION Field

Improved Multiplex Imaging of Targets in a Sample Employing StrandDisplacement.

BACKGROUND

DNA strand displacement is a method for the isothermal and dynamicexchange of DNA complexes.¹⁻³ Strand displacement can be designed andintentionally controlled based on an understanding of DNA hybridizationinteractions and thermodynamics, and can be facilitated by introducingengineered handles which are known as “toehold domains.” The ability tomodulate binding interactions and exchange hybridization partners givesrise to a series of potential applications. Several of theseapplications have been proposed and demonstrated by Diehl andcoworkers,⁴⁻⁷ including multiplexed protein detection and signalamplification applications in fixed cells using immunofluorescencemicroscopy.

The current state of the art as proposed by Diehl and coworkers providesonly a narrow implementation of strand displacement for fluorescencemicroscopy. The design proposed by Diehl and coworkers can be costlysince it requires conjugation of distinct nucleic acid sequences to eachtarget-recognition element in the assay. The efficiency of stranddisplacement is not optimal, and may induce unwanted non-specificbinding due to the use of longer DNA strands.

Here we propose several new, unanticipated embodiments based on DNAstrand displacement.

SUMMARY

In accordance with the description, a method for imaging of at least onetarget in a sample comprises: (a) providing at least onetarget-recognizing antibody bound to an adapter strand oligonucleotide;(b) providing a bridge strand oligonucleotide with a region capable ofspecifically binding the adapter strand oligonucleotide and a dockingstrand region; and (c) providing an imager strand oligonucleotidecapable of specifically binding the docking strand region of the bridgestrand oligonucleotide, wherein the imager strand is labeled with adetectable label.

In some embodiments, a method for imaging of at least one target in asample comprises (a) providing at least one target-recognizing antibodybound to a docking strand oligonucleotide; (b) providing a blockerstrand that hybridizes to the docking strand over a first region, butnot over its full length or the full length of the docking strand; (c)providing an imager strand oligonucleotide that hybridizes to thedocking strand oligonucleotide at least in the region of the dockingstrand that does not hybridize to the blocker strand, wherein the imagerstrand is labeled with a detectable label; (d) allowing the imagerstrand to hybridize to the docking strand, displacing the blockerstrand; (e) determining whether the target-recognizing antibody hasbound to the target.

In some embodiments, a method for imaging of at least two target in asample comprises (a) providing at least two target-recognizingantibodies bound to a docking strand oligonucleotide; (b) providing afirst imager strand oligonucleotide capable of hybridizing to thedocking strand on the first target-recognizing antibody and providing afirst blocking strand capable of hybridizing to the docking strand onthe second target-recognizing antibody, wherein the imager strand islabeled with a detectable label and the blocking strand is not labeledwith a detectable label and wherein optionally additional blockingstrands are used if more than two target-recognizing antibodies arepresent; (c) imaging the first target and removing the imager strand andblocking strand with at least one bouncer strand; (d) providing a secondimager strand oligonucleotide capable of hybridizing to the dockingstrand on the second target-recognizing antibody and providing a secondblocking strand capable of hybridizing to the docking strand on thefirst target-recognizing antibody, wherein the imager strand is labeledwith a detectable label and the blocking strand is not labeled with adetectable label and wherein optionally additional blocking strands areused if more than two target-recognizing antibodies are present; (e)imaging the second target; and (f) optionally removing the imager strandand blocking strand with at least one bouncer strand and optionallyrepeating the imaging for any additional targets.

In some embodiments, a method for imaging of at least one target in asample comprises: (a) providing at least one first target-recognizingantibody bound to a first docking strand oligonucleotide; (b) providingat least one second target-recognizing antibody bound to a seconddocking strand oligonucleotide; (c) providing an imager strandoligonucleotide capable of specifically binding to the first dockingstrand oligonucleotide and the second docking strand oligonucleotidewhen the first docking strand oligonucleotide and the second dockingstrand oligonucleotide are in proximity to each other forming afull-length docking strand, wherein the imager strand does notspecifically bind either the first or second docking strandoligonucleotide alone; (d) detecting the imager strand, wherein theimager strand is labeled.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice. The objects and advantageswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) andtogether with the description, serve to explain the principles describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the use of a universal reagent to link the docking strandto the target. 101 represents an antibody capable of binding to thetarget. The combination of 102 and 103 represent a universal adaptor,wherein 102 is an antibody binder and 103 is a universal adapter. Thecombination of 104-105 represent a bridge strand with a domain that iscomplementary to the universal adaptor (103) and a docking site (105).

FIG. 2 illustrates the use of a dynamic complex to introduce and removeimager strand using 4-way branch migration. The target binding agent ofFIG. 2 may be an antibody in some embodiments.

FIG. 3 illustrates one reaction (a) to introduce the imager strand andthe reaction (b and c) to remove it from the docking strand.

FIGS. 4A-D shows various embodiments of the use of blocker strands. FIG.4A shows three primary antibodies bound to their three respectivetargets and having three docking strands. In FIG. 4B, the sample istreated with fluorescent-labeled imager strand 210, complementary todocking strand 207. Unlabeled blocker strand 211 and 212, complementaryto docking strands 208 and 209, respectively, are added, before washingand imaging of target 204. Between the views shown in FIGS. 4B and 4C, amixture of bouncer strands complementary to the imager or blockerstrands 210, 211, and 212 are added to remove these strands. FIG. 4Dshows imaging of target 205 using fluorescently-labeled imager strand214 and blocker strands for the other docking strands.

FIG. 5 illustrates exchange imaging using blocker and bouncer strands.Binding domains are labeled by letters and letters with asterisks toindicate complementary pairs (e.g. domain a hybridizes to domain a*).First, all targets are stained with nucleic acid-target recognitioncomplexes, where the docking strand may contain two domains (e.g., a andb). Blocker strands for all targets can be introduced, where eachblocker strand is complementary to at least one domain of a dockingstrand (e.g., b*). Then, imager strands for the target(s) being imagedcan be introduced and will displace the blocker strand only for thetarget(s) to be imaged in the current imaging round. After acquiringimages, the bouncer strand containing domains complementary to theimager strands (e.g. a, b, and c) are introduced to displace and washaway the imager strands. This cycle can be repeated for additionalrounds of imaging of other targets for sequential multiplexing.

FIG. 6 shows exchange imaging using semi-universal blocker and universalbouncer strands.

FIG. 7 provides a method for multiplexed proximity detection with stranddisplacement of imager strands.

DESCRIPTION OF THE EMBODIMENTS I. Definitions

Docking strands are defined as oligonucleotides which are attached totargets of interest, in some embodiments, through an intermediatemolecule (e.g., antibody including full length antibodies and antigenbinding fragments thereof) to form a nucleic acid-target recognitioncomplex. A docking site is defined as a region of complementarity orspecific binding affinity between the docking strand and the imagerstrand.

Imager strands are defined as oligonucleotides attached to a label(e.g., fluorophore, nanoparticle, etc.). Blocker strands are defined asoligonucleotides with regions of complementarity to docking strandsthat, when bound to a docking strand, block association with anyoff-target imager strands; blocker strands likewise can beoligonucleotides with regions of complementarity to imager strands that,when bound to an imager strand, block association with any off-targetdocking strands (see FIG. 2). Bouncer strands are defined asoligonucleotides with regions of complementarity to blocker or imagerstrands that result in strand displacement of blocker or imager strandsthat are bound to docking strands.

When hybridization or hybridization conditions are referenced throughoutthe application, high ionic strength buffer conditions (e.g. 1× salinesodium citrate buffer, or 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, or 600mM sodium chloride in phosphate buffer) may be employed at roomtemperature to ensure hybridization occurs.

II. Universal Reagents to Link the Docking Strand to the Target

The Docking Strand is usually linked to the target via atarget-recognizing molecule such as an antibody (including antigenbinding fragments thereof) or an aptamer. Antibody is a popular type oftarget-recognizing molecule. Many antibody-DNA conjugation methods havebeen described in the past, including ones only involving covalent bondsand ones involving non-covalent bonds. Examples of conjugation methodsinvolving non-covalent bonds include ones involving naturally occurringand engineered antibody-binding proteins, such as Protein A, Protein Gand Protein A/G, as well as secondary antibodies and antibody fragments(e.g. Fab, Fab′, F(ab′)₂) that recognize the constant region/domains(e.g., Fc, CH1, CH2, CH3, CL domains).

To simply the procedure in attaching different Docking Strands (ofdifferent sequences) to different target-recognizing molecules (e.g.,antibodies) we here describe the use of one or a few universal adaptors.As shown in FIG. 1, one may conjugate a DNA strand with a ‘universal’(i.e., target-independent) adaptor sequence (103) to an antibody binder(102) that can bind a constant region/domain of primary antibody (101).The antibody binder (102) can be Protein A, Protein G, or the like, ormonovalent Fab, Fab′, or the like.13 Such universal adaptor (comprising102 and 103) can be complexed with the primary antibody by simpleincubation. With or without purification, one can then add a ‘bridgestrand’ to the complex. The bridge strand comprises two domains: one(104) complementary to the universal adaptor (103), and the other (105)serving as the docking site. The new, three-part complex can be usedwith or without purification. Optionally, one may add to the complexscavenger molecules that inactivate universal adaptor or bridge strandin excess to prevent unwanted interactions. For example, if the primaryantibody is I derived from mouse, one may use normal mouse IgG (ornormal mouse serum) to quench the excess universal adaptor (102-103) sothat the universal adaptor does not bind other mouse primary antibodiesthat may be present in the sample. For another example, one may usestand-alone DNA strand 103 to quench the excess bridge strand (104-105)so that the bridge strand does not bind universal adaptor strand boundto other targets.

One may also exercise part of this concept. For example, one maycovalently conjugate the universal adaptor sequence (103) to the primaryantibody (instead of using the antibody-binder, 102) to get the benefitof not having to chemically conjugate different DNA sequences to theantibody if, for example, the sequence of the docking site needs to beoptimized.

III. Use of Direct Hybridization to Introduce Imager Strand

In the demonstration of Diehl and colleagues, the Imager Strand isintroduced to the sample (which contains the Docking Strand as asingle-stranded DNA) as a part of a duplex that also contains a‘protector’ strand with sequence complementary to the Imager Strand. Theprotector strand ‘shields’ some bases on the Imager Strand and mayprevent it from interacting with non-target molecules via unwantedWatson-Crick pairing or other nonspecific interactions. However, thisdesign requires that the Imager Strand is long. To be specific, in theprior design, the portion of the Imager Strand that is complementary tothe docking site has to be long because it must contain two domains: (a)a single-stranded toehold domain that initiates the interaction with thedocking site, and (b) a domain that is bound to the protector strand.The toehold of (a) must be at least ˜5-nt-long (assuming ˜50% GCcontent) to initiate fast strand displacement, and the protector-bounddomain of (b) must be at least ˜12-nt-long (assuming ˜50% GC content) toensure stable hybridization. Therefore, in the prior design, the totallength of the portion of the Imager Strand that is complementary to thedocking site has to be at least ˜17-nt (assuming ˜50% GC content). As aresult, in the prior design, the docking site also has to be at least˜17-nt (assuming ˜50% GC content).

In our new design, the Imager Strand can be introduced as asingle-stranded DNA without the protector strand. In this design, thelength requirement of the portion of the Imager Strand that iscomplementary to the docking site is long enough to ensure stablehybridization with the Docking Strand, in some embodiments with theminimum length being ˜12-nt (assuming ˜50% GC content). The docking sitecan also be of similar length. The shorter length can make the designmore compact and reduce the chance of DNA-induced non-specific binding.

IV. Use Other Dynamic Complex to Introduce and Remove Imager Strand

A standard way to carry out toehold-mediated strand-displacement is touse 3-way branch migration involving a common strand, an incumbentstrand, and an invading strand. An example of this process is shown inthe ‘label’ reaction of FIG. 2A of US2016/0002704 A1, where thefluorescent-labeled strand is the common strand, the ‘waste’ strand isthe incumbent strand, and the NA tag is the invading strand. A drawbackof 3-way branch migration is that the invading strand must besingle-stranded. Such a long (at least ˜17-nt as described before)single-stranded DNA may cause unwanted non-specific binding.

As a new approach, to reduce the length of single-stranded DNA one mayuse 4-way branch migration to introduce and remove the Imager Strand.One example is shown in FIG. 2. FIG. 2 shows a docking strand attachedto a target-recognizing antibody with a docking strand 1 whichhybridizes over part of its length to a blocker strand 3 and over atleast some of the remaining length to imager strand 2 with an observablelabel. Imager strand 2 also hybridizes over part of its length to ablocker strand 4. This allows imager strand 2 to replace blocker strand3. Optionally, blocker strand 3 and blocker strand 4 hybridize to eachother over their full length. This embodiment shows both blocker strandsto docking strands and blocker strands to imager strands.

Another way to simplify the DNA constructs is to use hairpins. Forexample, one may use the reaction (a) of FIG. 3 to introduce the ImagerStrand (implemented by H1) and use the reaction (b and c) to remove itfrom the Docking Strand (implemented by C1). FIG. 3 is adapted fromNucl. Acids Res. (2011) 39 (16): e110.

V. Other Forms of Signal Amplification

One may replace the fluorophore (that is brought to the target via theDNA complexes), by other molecule or moieties that can be directly orindirectly observed. These molecules or moieties include, but are notlimited to, metal particles, plasmonic enhancers, enzymes (e.g., HRP),primer, capture oligonucleotide or splint of rolling circleamplification, initiator of finite or infinite hybridization chainreaction, etc.

VI. Use of Blocker Strands and Universal/Automated Exchange

A. Blocker Strands

Multiplexed imaging can be carried out with efficient exchange (e.g. viastrand displacement) of imager strands from one imaging round to thenext. For example, one may image the first target with the first imagerstrand, then displace the first imager strand with a washing step, thenintroduce a second imager strand to image a second target, and so on.One aspect is the addition of blocker strands for any targets that arenot actively being imaged in order to reduce cross-reactivity andprevent imager strands binding to off-target docking strands. Blockerstrands must be specific to one or more domain(s) of the docking strand,which is complexed to a specific target through a target-recognitionentity. In one embodiment, the blocker strand is bound to a portion ofthe docking strand such that a toehold domain that is complementary tothe imager strand remains unhybridized. Using this composition, animager strand that is complementary to multiple domains of the dockingstrand including, but not limited to the toehold domain, can beintroduced to displace the blocker strand prior to imaging the target.In another aspect, a bouncer strand can be introduced to strip theimager strand off of the docking strand. FIGS. 4A-D and FIG. 5demonstrate embodiments of the components and methods used formultiplexed imaging with the addition of blocker and bouncer strands forexchange.

One embodiment is a method for universal exchange of imager strands andreplacement of blocker strands to streamline sequential rounds ofimaging. In one method, one or more targets are detected with one ormore rounds of imaging wherein a cocktail of all bouncer strands or allblocker strands are introduced as intermediary steps between imagingrounds. A composition involving a mixture of all types of bouncerstrands is contemplated as a washing buffer to displace any bound imagerstrands. A composition involving a mixture of all types of blockerstrands is contemplated as an additive step prior to the addition of anyimager strands. This composition could be part of a kit. This methodcould be manual, or partially or fully automated. Fluidics could beincorporated for liquid handling. Software could be incorporated forimage acquisition and analysis. This method could be applied toimmunohistochemistry and immunofluorescence on cell or tissue samples.

Another way to achieve similarly simplified workflow and reduce crosshybridization among DNA species is to use mixtures comprising ImagerStrand(s) and Blocker Strand(s). Specifically, to image one target, onecan mix the Imager Strand for this target and the Blocker Strands forall other target and apply them to the sample containing all DockingStrands. Even if the Imager Strand incorrectly binds to the DockingStrand on a wrong target, it can be displaced by the Blocker Strand forthat target. One example is shown FIG. 4D. In this example we supposethere are 3 primary antibodies (201, 202, 203) that are bound to 3targets (204, 205, 206), and are attached with 3 Docking Strands (207,208, 209), respectively. After staining the sample with 3 DNA-attachedantibodies (Stage A), one can add a mixture of (i) fluorescent-labeledImager Strand 210, which is complementary to Docking Strand 207, (ii)unlabeled Blocker Strand 211, which is complementary to Docking Strand208, and (iii) unlabeled Blocker Strand 212, which is complementary toDocking Strand 209. After washing one can image target 204 (Stage B).Next, one can add a mixture of Bouncer strands that are complementary toImager/Blocker Strands 210, 211 and 212 to remove these strands from thesample (Stage C). Next, one can add a mixture of (i) unlabeled BlockerStrand 213, which is complementary to Docking Strand 207, (ii)fluorescent-labeled Imager Strand 214, which is complementary to DockingStrand 208, and (iii) unlabeled Blocker Strand 212, which iscomplementary to Docking Strand 209. After washing one can image target205 (Stage D). One can use a similar process visualize target 206.

B. Use of Semi-Universal or Universal Blocker Strands

A composition for a semi-universal blocker strand is described. Such asemi-universal blocker strand may contain one domain that is universal(i.e. constant across all docking strands complexed to all targets) andone domain that is target specific.

A composition for a universal bouncer strand is described. A universalbouncer strand reduces the complexity of finding unique sequences topair with each type of docking strand; and thus, may facilitateultra-high multiplexing. In one embodiment, the universal bouncer strandis composed of one domain that binds to a region of a docking strandthat is held constant for all target complexes. Binding of the universalbouncer strand may occur through toehold mediated strand displacementand may displace a semi-universal blocker strand.

A method for reiterative imaging and sequential multiplexing withsemi-universal blocker strands and universal bouncer strands isdescribed (FIG. 6). First, all targets are stained nucleic acid-targetrecognition complexes, where the complex includes a docking strand thatmay contain a constant domain (in FIG. 6 labeled as u*) and atarget-specific domain (in FIG. 6 labeled as N* for the Nth target).Semi-universal blocker strands (e.g. composed of a constant domain u,and a target specific domain N) for all targets can be added and bind tothe constant and target-specific domains of a docking strand. Then, amixture can be added containing the universal bouncer strands, imagerstrand for the Nth target, and all semi-universal blocker strandsexcluding any containing a domain specific to the Nth target. Duringthis step, the universal bouncer binds to a toehold available in theconstant region of the docking strand and begins to displace the blockerstrand. The imager strand binds to a toehold available in domain N* ofthe docking strand to complete the displacement of the blocker strand.The cocktail of semi-universal blocker strands for all other targets isadded to ensure those targets remain blocked. Then, image acquisitioncan be carried out to detect the Nth target. Once image acquisition iscomplete, a mixture of all semi-universal blocker strands, including ablocker strand specific to the Nth target, can be used to displace allimager strands and universal bouncer strands prior to the next cycle orround of imaging. This cycle can be repeated for additional rounds ofimaging of other targets for sequential multiplexing. It must beappreciated that the thermodynamics of each binding pair must beprecisely understood, and the equilibrium for each forward reactionprecisely controlled to facilitate a method involving a universalbouncer strand and semi-universal blocker strand.

VII. Proximity Detection

In another embodiment, a method of multiplexed proximity detection isproposed. Proximity detection is accomplished using a pair of nucleicacid-target recognition complexes, each of which contain a domaincorresponding to a partial (such as a half of a) docking site for animager strand. When the pair of nucleic acid-target recognitioncomplexes are in proximity to each other, the full docking site isformed allowing an imager or blocker strand to bind. The distancebetween target-recognition complexes can vary based on the design of thedocking strand, which can vary in length from 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 50, 100 or more nucleotides. Proximitydetection can be designed for distance lengths of 5 nm, 10 nm, 15 nm, 20nm, 30 nm, 50 nm, 100 nm, or more. High ionic strength buffer conditions(e.g. 1× saline sodium citrate buffer, or 150 mM, 200 mM, 300 mM, 400mM, 500 mM, or 600 mM sodium chloride in phosphate buffer) may beemployed at room temperature to ensure hybridization occurs. The pair ofnucleic acid-target recognition complexes can be designed to bind to asignal molecular target or two separate targets. An imager strand maycontain two domains, one domain complementary to the full docking site,and one domain that serves as a toehold for strand displacement.

For example, in FIG. 7, a target is bound with two DNA-conjugatedantibodies that bind to different epitopes on the same target, bringingthe DNA strands in proximity to form the full docking site. When thecorresponding imager strand is added, the imager strand will bind to thefull docking site, and the target can be imaged (e.g. withepi-fluorescence, confocal microscopy, TIRF, etc.). To extinguish theimaging signal, the imager strand can be displaced by the addition of abouncer strand which binds to a toehold on the imager strand.Multiplexed proximity detection of multiple targets can be carried outwith one or more rounds of strand displacement.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. The foregoingdescription and Examples detail certain embodiments and describes thebest mode contemplated by the inventors. It will be appreciated,however, that no matter how detailed the foregoing may appear in text,the embodiment may be practiced in many ways and should be construed inaccordance with the appended claims and any equivalents thereof.

As used herein, the term about refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term about generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited range) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). When terms such as at leastand about precede a list of numerical values or ranges, the terms modifyall of the values or ranges provided in the list. In some instances, theterm about may include numerical values that are rounded to the nearestsignificant figure.

REFERENCES

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1-13. (canceled)
 14. A method for imaging of at least one target in asample comprising: a. providing at least one target-recognizing antibodybound to a docking strand; b. providing a first blocker strand thathybridizes to the docking strand over a first region, but not over thefull length of the docking strand; c. providing an imager strand thathybridizes to the docking strand at least in the region of the dockingstrand that does not hybridize to the blocker strand, wherein the imagerstrand is labeled with a detectable label; d. allowing the imager strandto hybridize to the docking strand, displacing the blocker strand; e.imaging the at least one target; f. removing the imager strand from thedocking strand by employing a bouncer strand to displace the imagerstrand; g. optionally repeating steps a-f or any subset thereof.
 15. Themethod of claim 14, wherein the bouncer strand is a universal bouncerstrand capable of hybridizing to all of the docking strands if multipletargets are being imaged.
 16. The method of claim 14, wherein thebouncer strand is a universal bouncer strand capable of hybridizing toall of the imager strands if multiple targets are being imaged. 17-22.(canceled)
 23. The method of claim 14, wherein the docking strand has afirst and second region, wherein the first region is closer to theantibody, and the blocker strand hybridizes to the second region. 24.The method of claim 14, wherein if multiple targets are being imaged,the blocker strand is a universal blocker strand capable of hybridizingto a portion of each docking strand.
 25. The method of claim 14, whereinthe imager strand has a first, second, and third region and wherein thefirst and second region of the imager strand hybridize to the dockingstrand and wherein the first region hybridizes to the portion of thedocking strand that is not hybridized to the blocker strand.
 26. Themethod of claim 25, wherein the bouncer strand hybridizes to all threeregions of the imager strand.
 27. The method of claim 14, wherein ifmultiple targets are being imaged, a mixture of all blocker strands forthe multiple targets are provided in step b prior to providing anyimager strand.
 28. The method of claim 14, wherein if multiple targetsare being imaged, a mixture of all bouncer strands for the multipletargets are provided in step f to displace all bound imager strands. 29.The method of claim 14, wherein the label is a fluorophore.
 30. Themethod of claim 14, wherein the label is a metal particle, plasmonicenhancer, enzyme, primer, capture oligonucleotide or splint of rollingcircle amplification, or initiator of finite or infinite hybridizationchain reaction.
 31. The method of claim 14, wherein the sample is a cellor tissue.